Hum. Reprod. Advance Access originally published online on October 11, 2007
Human Reproduction 2007 22(12):3241-3248; doi:10.1093/humrep/dem323
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Functional analysis of the human inhibin 
subunit variant A257T and its potential role in premature ovarian failure
1 Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia 2 Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
3 Correspondence address. Tel: 613-9594-3221; Fax: 613-9594-6125; E-mail: ashwini.chand{at}princehenrys.org
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Abstract |
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BACKGROUND: A nucleotide substitution in the inhibin
subunit (INHA 769G>A, A257T) has been associated with premature ovarian failure (POF). We hypothesize this mutation causes a reduction in inhibin bioactivity, removing its suppression on the pituitary FSH secretion. The aim of this study is to establish if A257T inhibin has reduced bioactivity.
METHODS: Mouse L
T2 pituitary gonadotrope, human granulosa (COV434) and human embryonic kidney (HEK293) cells were co-transfected with an activin-responsive reporter and increasing amounts of wild-type or variant A257T inhibin subunit, and the degree of inhibin antagonism of activin signalling determined.
RESULTS: A 5-fold inhibition was observed with wild-type inhibin subunit overexpression (P < 0.001) (confirmed in HEK293 cells), while the A257T inhibin showed no inhibitory activity. In human ovarian COV434 transfected cells, while wild-type and A257T inhibin A had similar bioactivities, there was a significant reduction in the bioactivity of A257T inhibin B compared with wild-type inhibin B (P < 0.005). In all the three cell systems, overexpression of wild-type and A257T subunit resulted in a 2- to 6-fold increase in secretion of dimeric inhibin indicating the reduced inhibin response was not due to a failure of dimerization.
CONCLUSIONS: This study supports the hypothesis that the INHA 769G>A variant may increase susceptibility to POF with impaired inhibin B bioactivity and provides insight into the complex aetiology of POF.
Key words: inhibin A/inhibin B/inhibin in vitro bioactivity/premature ovarian failure/activin
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Introduction |
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Premature ovarian failure (POF) is a common clinical condition that affects 1% of all women and is characterized by a loss of ovarian function. POF is a highly heterogeneous condition, with numerous causes including autoimmune (Luborsky et al., 2000
) or genetic (Mattison et al., 1984) disorders, permanent damage to the ovaries (following chemotherapy, irradiation or surgery) or exposure to environmental toxicants (Matikainen et al., 2001). However, the basis for the majority of POF cases remains unknown. Approximately, 45% of all POF cases can be attributed to genetic causes (Davis et al., 2000), with disorders linked to the X chromosome such as Fragile X and Turner’s syndrome giving rise to the POF phenotype (Conway et al., 1995), as do deletions at critical regions on Xq13.3–q21.1 (Sala et al., 1997) and Xq26–28 (Delon et al., 1997). In familial POF cases, inheritance suggests autosomal dominant or X-linked inheritance, characterized by incomplete penetrance (Mattison et al., 1984; Vegetti et al., 1998).
Recent studies have identified genetic mutations known to cause POF in the FSH receptor, (Aittomaki et al., 1996; Beau et al., 1998; Doherty et al., 2002; Meduri et al., 2003), FSH subunit (Matthews et al., 1993; Layman et al., 1997a,b), FOXL2 (Crisponi et al., 2001; Harris et al., 2002; Schlessinger et al., 2002), bone morphogenetic protein (BMP)15 (Di Pasquale et al., 2004; Chand et al., 2006; Dixit et al., 2006b; Laissue et al., 2006) and growth differentiation factor (GDF) 9 (Dixit et al., 2005; Chand et al., 2006; Laissue et al., 2006) genes.
A nucleotide substitution in the inhibin subunit gene (INHA 769G>A) was first identified in a New Zealand population (Shelling et al., 2000). The heterozygous 769G>A change, which results in the substitution of the alanine by a threonine residue at codon 257 (A257T) was significantly greater in women with POF (7.0%, n = 43) compared with controls (0.7%, n = 150, P = 0.010). Similar findings have been reported in two other populations (Marozzi et al., 2002; Dixit et al., 2004, 2006a). However, a mutational screening study in a Korean POF population did not identify any INHA 769G>A carriers (n = 84) (Jeong et al., 2004). More recently, a study in an Argentinean population showed no association between the INHA 769G>A variant and POF (POF 1.7%, n = 59, controls > 40 years 2.6%, n = 76) (Sundblad et al., 2006). All of the above studies were performed on small numbers of patients, posing the question of whether this association is significant, or whether the varying frequencies in different populations are due to ethnic dependent effects.
Currently, there is no functional evidence to confirm the physiological role of the INHA 769G>A variant in the pathogenesis of POF. It is well established that inhibin is a secreted product of the ovary that regulates pituitary FSH synthesis and secretion thereby promoting ovarian folliculogenesis. In addition, inhibin has been implicated in the direct intraovarian regulation of folliculogenesis by its role as an antagonist to GDF9 (Wu et al., 2004). The mode of inhibin action, particularly in the pituitary, is based on its inhibition of the action of activin, a known stimulator of FSH secretion (Robertson et al., 1986; Vale et al., 1986). Owing to the endocrine and paracrine actions of inhibin, it is hypothesized that the mutated form, being less biologically active, would contribute to POF (Shelling et al., 2000).
To test the hypothesis that the A257T inhibin variant is less bioactive than the wild type, inhibin’s antagonism of an activin-responsive promoter (p3XGRAS) in three cell lines is examined. We conclude that the A257T inhibin is less active than its wild-type form in antagonizing activin A/B-dependent reporter gene expression and that the A257T inhibin B variant (but not inhibin A) has a reduced bioactivity. These data support our hypothesis that the A257T inhibin may contribute towards the POF phenotype.
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Materials and Methods |
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Cloning of inhibin expression vectors
The two INHA nucleotide variants (769G and 769A) were amplified from patient DNA using PCR with Expand HiFidelity Taq polymerase. The amplified products were cloned into the pCDNA3.1 expression vector and transformed into DH5
TM competent cells (Invitrogen, Australia). Ampicillin resistant colonies were selected, and plasmid DNA was purified from cells using CONCERTTM Rapid Plasmid Miniprep System (Invitrogen) in accordance with the manufacturer’s instructions. DNA concentrations were measured at 280 nm absorbance. Mutant and wild-type clones were confirmed by DNA sequencing.
Cell culture
Cell lines used for this study included the LT2 mouse pituitary gonadotrope (Windle et al., 1990), human ovarian granulosa cell carcinoma (COV434) (Nishi et al., 2001) and human embryonic kidney (HEK293) cell lines. Cells were maintained in culture in the absence of antibiotics in 175 cm2 culture flasks at 37°C in 5% CO2 in a humidified incubator. The culture medium consisted of Dulbecco’s modified Eagle’s medium (4.5 g/l glucose), buffered with sodium bicarbonate (37 mg/l) and 25 mM HEPES; supplemented with 10% fetal calf serum (FCS) (all obtained from Trace Biosciences, Melbourne, Australia) and sodium pyruvate (110 mg/l) (Sigma, Australia).
Transfections
Cells (20 000 cells/well) were plated in 48-well plates and transfections carried out on Day 2 with a DNA:lipofectamine ratio of 1:3 (Invitrogen). The reporter constructs contained activin stimulatory regions consisting of three copies of the GnRH receptor activating sequence (3XGRAS) (Duval et al., 1997) used to drive expression of the luciferase reporter gene. The reporter construct p3XGRAS-PRL-Luc was a gift from Dr B.S. Ellsworth (Colorado State University, Collins, USA). In LT2 cells, each well was transfected with 0.25 µg of the reporter construct plus inhibin subunit (wild-type and variant) vectors at various amounts up to 1.0 µg DNA. In all transfection wells, a total of 1.0 µg DNA was transfected, the balance of DNA being adjusted with the addition of the empty plasmid vector (pCDNA3.1). In COV434 cells, the varying amounts of subunit were co-transfected with 0.025 µg of A- or B-containing plasmids. The transfected cells were incubated in culture medium containing the DNA–Lipofectamine complexes for 24 h. The following day, culture media were replaced with media containing 0.2% FCS, with or without activin A or B (0.2 nM, R&D Systems, Minneapolis, USA) and incubated for 24 h. The culture media were collected and stored at –80°C.
In initial experiments, co-transfection of a -galactosidase reporter construct was used for normalization of transfection data. This gave inconsistent luciferase readings, with levels of -galactosidase activity inversely proportional to the amount of inhibin produced, due most likely to the observation that -galactosidase activity is stimulated by activin (data not shown). Similar results have been reported by others (Pangas et al., 2002). Therefore, transfection efficiency was monitored on the basis of inhibin protein levels in culture media.
In HEK293 cells, another activin-sensitive reporter, pA3-Lux reporter gene, which has three tandem copies of the activin-responsive element from the Xenopus Mix2 gene was used (Nagarajan et al., 1999). As HEK293 cells lack the Smad-interacting forkhead transcription factor FAST2, essential for the activin signalling pathway, wild-type and mutant inhibin subunit plasmids were co-transfected with the pA3-Lux reporter construct and pFAST. Data for transfection experiments are presented as the mean ± SD of triplicates from one representative experiment. All experiments shown were repeated at least three times.
Luciferase assays
Transfected cells were lysed with lysis solution [250 µl, 1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol (DTT)]. For the luciferase assay, cell lysate (100 µl) and assay buffer (350 µl, 15 mM potassium phosphate buffer, pH 7.8, containing 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 2 mM ATP) were mixed and luciferase activity measured for 1 s using a luminometer (Berthold Australia, VIC, Australia) after an addition of the luciferase substrate (20 mM Luciferin, 25 mM glycylglycine and 2 mM DTT) (Promega Corp. Madison, WI, USA). The luciferase assay results are presented as a fold change relative to basal promoter activity.
Quantitation of inhibin
Transfection efficiency of every experiment was gauged by the measurement of inhibin levels in the conditioned media of transfected cells. Inhibin A and B were quantitated by specific enzyme-linked immunosorbent assays (ELISAs) (DSL, Webster, TX, USA), provided by Oxford Bio-innovation Ltd (Upper Heyford, UK) and used according to manufacturer’s specifications. The sensitivity and inter-assay variation for the inhibin A ELISA were 6 pg/ml and 10%; for inhibin B ELISA: 3 pg/ml and 12%, respectively.
Cell proliferation assay
Assessment of cell density of transfected cells in vitro was measured with the CyQuant Cell Proliferation Assay Kit (Molecular Probes Inc., OR, USA) according to manufacturer’s instructions. Data are presented as the mean ± SD of triplicates from one representative experiment. The experiment was repeated three times.
Statistical analysis
Comparison of sample means from each transfection condition and activin treatment groups were analysed using paired Student’s t-test, whereas analysis of variance was used to compare data from three separate experiments. A value of P < 0.05 was considered significant.
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Assessment of the A257T
subunit variant bioactivityL
T2 gonadotrope cells act as an endogenous activin- and inhibin-responsive system, expressing both the activin receptors and betaglycan. To investigate the effect of the INHA 769G>A (A257T) subunit variant on inhibin biological activity, LT2 cells were co-transfected with an activin-sensitive luciferase construct (p3XGRAS-PRL-Luc) and increasing amounts of either wild-type or variant subunit. Transfection efficiency was assessed by the measurement of secreted inhibin B (LT2 cells express B but not the A subunits, data not shown) in the conditioned medium (Fig. 1C). The transfection of wild-type inhibin subunit resulted in a dose-dependent decrease in p3XGRAS-PRL-Luc activity, with a 5-fold decrease observed at the 0.6 µg dose (Fig. 1A). In contrast, activin-induced p3XGRAS-PRL-Luc activity was unaffected by transfection of increasing doses of the variant subunit complementary DNA (cDNA) (P < 0.001, Fig. 1A), indicating that the A257T variant inhibin B has compromised biological activity. To demonstrate the changes in reporter activity caused by wild-type and variant inhibin were not due to changes in cell number, cell proliferation rates were determined in transfected cells showing no significant dose-related effects (Fig. 1C).
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The decline in basal p3XGRAS-PRL-Luc reporter activity may be due to an inhibin-specific effect (i.e. acting directly to antagonize activin actions through receptor-mediated mechanisms) or a consequence of decreased endogenous activin levels resulting from increased
subunit dimerization (caused by forced expression of higher amounts of subunit) (Fig. 2). We therefore assessed whether transfection of either wild-type or variant subunit expression plasmids can similarly affect activin-stimulated promoter activity. Basal p3XGRAS-PRL-Luc reporter activity was elevated 15- and 3-fold following activin A and B stimulation, respectively (Fig. 2A). Similar to observations in untreated cells, a clear difference in the inhibin response was observed between cells transfected with wild-type and variant subunit cDNA following both activin A (P < 0.001) and activin B (P < 0.001) treatment (Fig. 2C and D, respectively). Dimeric wild-type and A257T variant inhibin B isoforms were expressed at similar levels (Fig. 2B) in transfected cells indicating equivalent transfection efficiencies.
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To further confirm the above findings, either wild-type or A257T
subunits were overexpressed in HEK293 cells, which unlike LT2 cells express inhibin A and B subunits. Luciferase activity was suppressed with wild-type inhibin subunit (5-fold) but not with A257T subunit overexpression, at 0.6 µg cDNA dose (P < 0.001, Fig. 2E).
Co-transfection of wild-type or variant with A or B subunits in COV434 cells
A decrease in the amount of bioactive inhibin, as postulated in women with the INHA A257T mutation, could also affect local ovarian function. Therefore, the COV434 human granulosa cell line was used to examine the effects of inhibin A and B isoforms separately. Basal inhibin A and B levels in conditioned media were detectable above the assay sensitivity, while co-transfection with wild-type or variant and A or B subunits yielded a 20-fold increase in inhibin A and B expression (Fig. 3B and D). Wild-type and A257T inhibin A demonstrated the same degree of suppression of p3XGRAS-PRL-Luc transcriptional activity (Fig. 3A). In contrast, a significant difference (P < 0.005) was shown in the suppression of p3XGRAS-PRL-Luc activity by the wild type but not with the A257T inhibin B (Fig. 3C). This data, along with the transfection studies in LT2 cells indicate the INHA 769G>A nucleotide change (resulting in the A257T subunit variant) significantly impairs inhibin B bioactivity. The COV434 data show that the A257T inhibin A shows no biological difference to wild-type inhibin A. Levels of mutant inhibin A measured in conditioned medium showed no difference compared to wild type (Fig. 3B).
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Discussion |
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The present study examines the functional significance of the INHA 769G>A (A257T) nucleotide change using in vitro assay systems. Transient protein expression of inhibin
subunit cDNA (wild type or variant) in mouse pituitary gonadotrope (LT2), human ovarian granulosa (COV434), and human embryonic kidney epithelial (HEK293) cells demonstrated reduced activin-antagonist activity of the A257T variant inhibin compared with wild-type inhibin forms.
In the transfection experiments, overexpression of the wild-type subunit led to an increase in dimeric inhibin and a dose-related inhibition of activin-stimulated reporter activity. The inhibitory action of inhibin is mediated by association with its co-receptor, betaglycan, followed by the formation of high affinity complexes with the activin type II receptors. The inhibin–betaglycan complex is thus a potent competitor of activin.
LT2 cells, which express both betaglycan and activin-signalling components (Pernasetti et al., 2001; Otsuka and Shimasaki, 2002; Dupont et al., 2003), are well characterized for assessment of activin and inhibin responses in vitro (Graham et al., 1999; Ethier et al., 2002). In the LT2 cells, the selective expression of inhibin B, but not the or A subunits, was an important feature aiding experimental design. Transfection of increasing amounts of inhibin subunit (wild-type and A257T variant) in LT2 cells and its subsequent dimerization with endogenous subunits corresponded to increased inhibin levels in the conditioned media. Using the inhibin ELISAs, detection of dimeric A257T variant inhibin proteins secreted into culture medium of transfected cells, showed that there was no significant impairment in the ability of the A257T subunit to dimerize with the A or B subunits.
Unlike the wild-type form, the A257T inhibin was unable to suppress activin-stimulated reporter activity as efficiently in LT2 and HEK293 cells suggesting that A257T mutant inhibin B is biologically less active. A possible explanation for this reduced bioactivity could be a lower affinity for betaglycan or the activin type II receptors. Our own results (Harrison, unpublished) have shown that the subunit alone is biologically inactive in a rat pituitary cell culture system and thus one would conclude that dimeric inhibin rather than the inhibin subunit is biologically functional.
Measurement of secreted inhibin A (in COV434 and HEK293 cells) and inhibin B (in LT2, COV434 and HEK293 cells) showed similar dose-related increases with wild-type and variant subunit overexpression, discounting the possibility that the loss of A257T inhibin B bioactivity is due to defective subunit dimerization. Furthermore, the addition of exogenous activin A and B, to transfected cells, did not alter the differences observed between wild-type and A257T inhibin B activity, substantiating that these differences are reflective of a significant difference in the inhibin-specific (activin antagonist) function.
Subsequent transfection studies in the ovarian cancer cell line, COV434 indicated that the A257T subunit variant had significantly less effect on the inhibin A response compared to the inhibin B response. These results raise the possibility that inhibin A and B may be functionally/mechanistically distinct. In support, Brown et al. (2000, 2003) recently showed that B subunit overexpression could rescue the inhibin A null embryonic lethal phenotype. In this study, mice were generated in which the mature region of the A subunit gene was replaced with the corresponding mature region of the B subunit gene. Although some of the defects of the A subunit null mice were rescued and the mice survived, they displayed a variety of defects including hypogonadism, decreases in body mass, life expectancy, female fertility and hair growth. These defects were attributed to the non-overlapping functions of A and B subunits.
Alignment of amino acid sequences across various mammalian species indicates that the alanine to threonine substitution caused by the INHA 769G>A nucleotide change occurs in a region which is highly conserved in many species including the mouse, bovine, porcine and equine sequences suggesting a regulatory function of this subunit region in inhibin bioactivity.
In conclusion, we demonstrate a functional consequence of the amino acid substitution at residue 257 on inhibin B bioactivity.
It is suggested that a decrease in inhibin B bioactivity could affect ovarian follicle reserve in a number of ways. It could provide a less potent negative feedback on the pituitary, leading to increased FSH levels, ovarian stimulation and aberrant folliculogenesis (Shelling et al., 2000). Recently, a transgenic mouse that overexpresses pituitary-independent FSH, a similar effect to the monotropic rise in FSH levels observed in ageing women, has been shown to have increased ovulation rates followed by infertility (McTavish et al., 2007). In this model, increased litter size and corpora lutea numbers reflect the increase in ovulation rates. Although no premature depletion of the follicle pool was observed, these mice experienced premature infertility after 26 weeks. This suggests a complex interaction of FSH and local ovarian factors in the regulation of folliculogenesis. Hence it is suggested that diminished bioactivity of inhibin would have a pronounced paracrine effect within the follicular microenvironment.
Several other animal studies implicate that a rise in FSH levels may contribute in the development of POF. Experiments where ewes and glits are immunized against inhibin result in increased ovulation rates with increased FSH release (Forage et al., 1987; Brown et al., 1990; O’Shea et al., 1993). In a mouse model, immunization against inhibin resulted in an autoimmune oophoritis and early ovarian failure (Altuntas et al., 2006). The proinflammatory phenotype in these mice was characterized by elevated production of interferon-
and IL-2. The increase in follicle numbers at all stages of follicular development including atretic follicles was due to the prolonged estrous cycles with the high FSH levels observed. The litter size was increased in the immunized mice due to ovarian stimulation. Interestingly in older inhibin immunized mice (43–45-week-old) the ovaries were atrophic with few viable follicles compared with age-matched controls. This study provides evidence that the suppression of inhibin and the resulting elevation of FSH levels cause ovarian stimulation, an earlier depletion of follicles and an accelerated rate of ovarian failure. The disparity in the ovarian phenotype between the FSH overexpressing mouse model (McTavish et al., 2007) and the inhibin immunized mice (Altuntas et al., 2006) may reflect an important role of inhibin in follicular maturation processes.
The mode of action for inhibin is via competition for activin and BMP type II receptors (ActRII and BMPRII) facilitated by betaglycan (Lewis et al., 2000; Wiater et al., 2003). Hence the paracrine effects of inhibin within the ovarian follicle are principally to antagonize activin and BMP action. The inter-relationship of inhibin with other transforming growth factor- superfamily members (such as GDF9, BMP15 and activin) highlights an important multifaceted paracrine/autocrine role for inhibin in the regulation of granulosa and thecal cell differentiation, antral formation and oocyte maturation and ovulation (reviewed in Findlay et al., 2002; Shimasaki et al., 2004).
Inhibin expression is localized in the developing gonads of the human foetus suggesting a role of inhibin in primordial follicle development (Eramaa et al., 1992). In the adult ovary, within the theca-intestitial cells, inhibin stimulates LH-dependent androgen production (Hsueh et al., 1987; Hillier and Miro, 1993) and enhances oocyte maturation and fertilization in vitro (Alak et al., 1996; Stock et al., 1997). A lower exposure to inhibin during foetal development in INHA G769A mutation carriers may impair the initial development of a healthy primordial follicle pool, possibly impacting granulosa cell proliferation and oocyte maturation processes. Thus, an in vivo mutant inhibin mouse model would be useful in further assessing whether POF is a physiological consequence of the inhibin A257T variant, and which of the two inhibin-responsive pathways predominates in the regulation of ovarian function.
The current study provides evidence that the INHA G769A mutation impairs inhibin B bioactivity and may be a contributing factor in the development of POF. In addition, the study provides important insights into the role of inhibin in folliculogenesis.
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Funding |
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NHMRC of Australia Program (#241000), Research Fellowship (DMR) (#169201), Auckland Medical Research Foundation and University of Auckland Research Committee Grants.
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Submitted on May 23, 2007; resubmitted on September 12, 2007; accepted on September 17, 2007.
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